Objective To investigate the effects of extracellular vesicles(EVs)derived from synovial fluid of rheumatoid arthritis(RA)patients on angiogenesis of human umbilical vein endothelial cells,and to preliminarily explore the underlying mechanisms.Methods Synovial fluid samples of knee joint were collected from 20 patients with RA and 20 patients with osteoarthritis(OA)in this study.EVs were purified using ultracentrifugation.The morphology and size of EVs were observed by transmission electron microscopy and nanoparticle tracking analysis.CD9,CD63,cytochrome c(Cyt-c),vascular endothelial growth factor(VEGF),lysyl oxidase(LOX),matrix metalloproteinase 2(MMP2),tumour necrosis factor alpha(TNF-α),transforming growth factor beta1(TGF-β1)in EVs were detected using Western blot.Human umbilical vein endothelial cells(HUVEC)were treated with the EVs.The growth,migration and angiogenesis of HUVEC were observed by CCK8 assay,TranswellTM chamber assay,scratch test and matrigel angiogenesis assay,respectively.The effect of EVs on the PI3K/AKT pathway in HUVEC was assessed using Western blot.Results Both EVs from RA synovial fluid(RA-EVs)and OA synovial fluid(OA-EVs)were cup-shaped,mainly between 30-400 nm in diameter,expressing CD63 and CD9,but not Cyt-c.RA-EVs carried more VEGF,LOX,MMP2,TNF-α and TGF-β1 than OA-EVs.Compared with the OA-EVs intervention,RA-EVs significantly promoted the proliferation,migration,and angiogenesis of HUVECs,as well as upregulated PI3K/AKT phosphorylation.The inhibitor of PI3K suppressed angiogenesis induced by EVs.Conclusion EVs in synovial fluid of RA carried more cytokines and enzymes that related angiogenesis and inflammation.These EVs exert their pro-angiogenic effects by activating the PI3K/AKT pathway,then contributing to the pathological progression of RA.

Objective To investigate the effects of synovial fluid from patients with rheumatoid arthritis(RA)on the polarization of normal neutrophils and the MEK/ERK signaling pathway,thereby providing experimental evidence to elucidate the pathological mechanisms of RA and offering novel insights into its treatment.Methods Synovial fluid samples were collected from 20 RA patients and 20 osteoarthritis(OA)patients.ELISA was used to measure the levels of interferon-gamma(IFN-γ),tumor necrosis factor-alpha(TNF-α),transforming growth factor-beta(TGF-β)and interleukin-8(IL-8)in the synovial fluid.Neutrophils were isolated from the peripheral blood of healthy donors,and their purity was confirmed by flow cytometry.Neutrophils were then treated with synovial fluid and divided into four groups:Control(untreated),RA(treated with 10％RA synovial fluid),OA(treated with 10％OA synovial fluid),and anti-IFN-β(treated with 20 μg anti-IFN-β antibody as an N2-type control).Western blot was used to assess the changes in the expression of IFN-γ,TNF-α,TGF-β,MEK,p-MEK,ERK and p-ERK.Additionally,the MEK inhibitor PD0325901 was used to block the MEK/ERK pathway,and subsequent changes in IFN-γ and TGF-β expression in neutrophils were evaluated.Results The levels of IFN-γ,TNF-α,TGF-β and IL-8 in the synovial fluid of RA patients were significantly higher than those in OA patients.After intervention with RA synovial fluid,the relative expression of IFN-γ and TNF-α in neutrophils were significantly increased compared to those in the untreated control group and the OA group.While,TGF-β expression in the RA group was lower than that in both the control and anti-IFN-β groups.The relative expression of p-MEK and p-ERK in the RA group were significantly higher than those in the control,OA and Anti-IFN-β groups.Upon addition of the MEK inhibitor,the relative expression of p-MEK and p-ERK were reduced.Furthermore,in both the RA and OA groups,the relative expression levels of IFN-γ decreased,while the expression of TGF-β increased.Conclusion Synovial fluid from RA patients contains higher of IFN-γ,TNF-αand TGF-β,which activate neutrophils through phosphorylation of the MEK/ERK signaling pathway,promoting their polarization toward the pro-inflammatory N1 phenotype.

Objective To investigate the effects of synovial fluid from patients with rheumatoid arthritis(RA)on the polarization of normal neutrophils and the MEK/ERK signaling pathway,thereby providing experimental evidence to elucidate the pathological mechanisms of RA and offering novel insights into its treatment.Methods Synovial fluid samples were collected from 20 RA patients and 20 osteoarthritis(OA)patients.ELISA was used to measure the levels of interferon-gamma(IFN-γ),tumor necrosis factor-alpha(TNF-α),transforming growth factor-beta(TGF-β)and interleukin-8(IL-8)in the synovial fluid.Neutrophils were isolated from the peripheral blood of healthy donors,and their purity was confirmed by flow cytometry.Neutrophils were then treated with synovial fluid and divided into four groups:Control(untreated),RA(treated with 10％RA synovial fluid),OA(treated with 10％OA synovial fluid),and anti-IFN-β(treated with 20 μg anti-IFN-β antibody as an N2-type control).Western blot was used to assess the changes in the expression of IFN-γ,TNF-α,TGF-β,MEK,p-MEK,ERK and p-ERK.Additionally,the MEK inhibitor PD0325901 was used to block the MEK/ERK pathway,and subsequent changes in IFN-γ and TGF-β expression in neutrophils were evaluated.Results The levels of IFN-γ,TNF-α,TGF-β and IL-8 in the synovial fluid of RA patients were significantly higher than those in OA patients.After intervention with RA synovial fluid,the relative expression of IFN-γ and TNF-α in neutrophils were significantly increased compared to those in the untreated control group and the OA group.While,TGF-β expression in the RA group was lower than that in both the control and anti-IFN-β groups.The relative expression of p-MEK and p-ERK in the RA group were significantly higher than those in the control,OA and Anti-IFN-β groups.Upon addition of the MEK inhibitor,the relative expression of p-MEK and p-ERK were reduced.Furthermore,in both the RA and OA groups,the relative expression levels of IFN-γ decreased,while the expression of TGF-β increased.Conclusion Synovial fluid from RA patients contains higher of IFN-γ,TNF-αand TGF-β,which activate neutrophils through phosphorylation of the MEK/ERK signaling pathway,promoting their polarization toward the pro-inflammatory N1 phenotype.

Objective To investigate the effects of extracellular vesicles(EVs)derived from synovial fluid of rheumatoid arthritis(RA)patients on angiogenesis of human umbilical vein endothelial cells,and to preliminarily explore the underlying mechanisms.Methods Synovial fluid samples of knee joint were collected from 20 patients with RA and 20 patients with osteoarthritis(OA)in this study.EVs were purified using ultracentrifugation.The morphology and size of EVs were observed by transmission electron microscopy and nanoparticle tracking analysis.CD9,CD63,cytochrome c(Cyt-c),vascular endothelial growth factor(VEGF),lysyl oxidase(LOX),matrix metalloproteinase 2(MMP2),tumour necrosis factor alpha(TNF-α),transforming growth factor beta1(TGF-β1)in EVs were detected using Western blot.Human umbilical vein endothelial cells(HUVEC)were treated with the EVs.The growth,migration and angiogenesis of HUVEC were observed by CCK8 assay,TranswellTM chamber assay,scratch test and matrigel angiogenesis assay,respectively.The effect of EVs on the PI3K/AKT pathway in HUVEC was assessed using Western blot.Results Both EVs from RA synovial fluid(RA-EVs)and OA synovial fluid(OA-EVs)were cup-shaped,mainly between 30-400 nm in diameter,expressing CD63 and CD9,but not Cyt-c.RA-EVs carried more VEGF,LOX,MMP2,TNF-α and TGF-β1 than OA-EVs.Compared with the OA-EVs intervention,RA-EVs significantly promoted the proliferation,migration,and angiogenesis of HUVECs,as well as upregulated PI3K/AKT phosphorylation.The inhibitor of PI3K suppressed angiogenesis induced by EVs.Conclusion EVs in synovial fluid of RA carried more cytokines and enzymes that related angiogenesis and inflammation.These EVs exert their pro-angiogenic effects by activating the PI3K/AKT pathway,then contributing to the pathological progression of RA.

Background and objective Treatment options for patients with squamous cell carcinoma of the lung (SCC) are limited in chemotherapy. However, not all patients could benefit form standard platinum regimen. Considering the dismal prognosis of patients with advanced SCC, a greater focus on selecting sensitive chemotherapy regimens remains of up-most importance to improve outcomes in this disease. In this study, we used matrix-assisted laser desorption/ionization time-of-flight mass spectrometry to detect pre-chemotherapy serum peptides in advanced lung squamous cell carcinoma patients accepting paclitaxel combined with platinum chemotherapy and to analyze the correlation between serum peptides and che-motherapy efcacy. Methods Patients with advanced lung squamous cell carcinoma received paclitaxel combining with plati-num chemotherapy and evaluated the efcacy every two cycles. Evaluation of complete response (CR) or partial response (PR) patients defined as sensitive group, progressive disease (PD) patients defined as resistant group. Serum samples were collected from patients with lung squamous cell carcinoma. Eighty-one patients were randomly divided into training group (sensitive group Ⅰ and resistant group Ⅰ) and validation group (sensitive group Ⅱ and resistant group Ⅱ) according to the ratio of 3:1. Se-rum samples were pretreated and Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was used to detect serum peptide fingerprints. ClinProTools software was used to analyze the differences between the sensitive group Ⅰ and the resistant group Ⅰ. Three kinds of biological algorithms (SNN, GA, QC) built in CPT software were used to establish the curative effect prediction model respectively and the optimal algorithm was selected. The validation group was used for blind verification. Results Thirty sensitive patients and 31 resistant patients were enrolled in the training group. Ten sensitive patients and 10 resistant patients were included in the validation group. The training group had 96 differentially expressed peptides in the sensitive and resistant patients, with 16 statistically significant peptides (P<0.001). The predictive model was established by 5 polypeptides (1,897.75 Da, 2,023.93 Da, 3,683.36 Da, 4,269.56 Da, 5,341.29 Da). The recognition rate of this model was 89.18% and the cross validation rate was 95.11%. The accuracy of the model was 85%, the sensitivity was 90.0% and the specificity was 80.0%. The median PFS in the sensitive group was better than patients in the resistant group (7.2 months 95%CI: 4.4-14.5 vs 1.8 months 95%CI: 0.7-3.5). The results showed that the differential peptides 4,232.04 Da and 4,269.56 Da were correlated with PFS in patients with lung squamous cell carcinoma (P<0.001). Conclusion MALDI-TOF-MS was used to detect the difference of serum peptides between sensitive and resistant groups. The preliminary curative effect prediction model was used to predict the efcacy of paclitaxel combined with platinum regimen. However, this model need further investigations to verify the accuracy and the sensitivity.

Background and objectivehTis study aimed at using matrix-assisted laser desorption ionization - time of lfight mass spectrometer (matrix-assisted laser desorption ionization time-of-lfight mass spectrometry, MALDI-TOF-MS) screening the difference serum peptides during epidermal growth factor tyrosine kinase inhibitors (EGFR-TKIs) treatment and exploring their signiifcance of advanced NSCLC patients.MethodsCollect 102 serum samples from 34 advanced NSCLC pa-tients, which are before TKI treatment, best effect of treatment and atfer progession. Peptides were extracted from the samples and then detected by MALDI-TOF-MS system to get the mass spectra. hTe mass spectra data was analyzed by the Clinpro-ToolTM sotfware to identify the different serum peptides, and then analyzed the clinical signiifcance of peptides.Results Among the 34 patients who received TKI treatment, there were none evaluated as complete response (CR), 11 patients evalu-ated as PR and 23 patients evaluated as stable disease (SD), with the PFS was 8.0 months (95%CI: 6.6-11.2); overall survival (OS) was 11.4 months (95%CI: 10.6-16.5). Atfer detected the serum from three different points of time, the result showed that they were totally different; 87 different peptide peaks were identiifed atfer analysis self-paired serum between the time of best effect and baseline, which included one statistically different [P<0.001, area under curve (AUC)≥0.9] peptide; 96 different peptide peaks were identiifed atfer analysis serum between the time of progression and baseline, which included 3 statistically different (P<0.001, AUC≥0.9) peptides; 115 different peptide peaks were identiifed atfer analysis serum between the time of progression and best effect, which included 4 statistically different (P<0.001, AUC≥0.9) peptides.ConclusionhTeserum peptides of NSCLC patients in the process of TKI treatment are dynamic and the different peptides may be associated with treatment effect and disease progression. However, the features and clinical signiifcance of different peptides need to be vali-dated in the future.

Background and objective Gemcitabine and taxanes are effective agents commonly used in advanced squamous lung cancer. hTe best treatment sequence, however, is unclear to our knowledge. So we conducted this retrospective study in order to compare the effcacy and toxicities of ifrst-line Gemcitabine+/-platinum followed by second-line taxanes+/-platinum with the reverse sequence. Methods We totally analyzed 105 patients with stage IIIb-IV squamous lung cancer in our retrospective study. hTere were 49 patients receiving gemcitabine+/-platinum ifrst-line followed by taxanes+/-plati-num second-line (G-T group), and 56 patients receiving taxanes+/-platinum ifrst-line followed by gemcitabine+/-platinum second-line (T-G group). hTe primary endpoint of the study was overall survival (OS), and the secondary endpoints included progression-free survival (PFS), objective response rate (ORR), disease control rate (DCR) and toxicities. Results hTe me-dian OS were 18.5 mo in G-T group and 19.0 mo in T-G group (P=0.520). hTe median PFS1 was 5.0 mo and 4.0 mo with ifrst-line gemcitabine+/-platinum and taxanes+/-platinum, respectively (P=0.584). hTe median PFS2 was 2.7 mo and 2.5 mo with second-line gemcitabine+/-platinum and taxanes+/-platinum (P=0.432). hTe ORR1 of G-T group and T-G group were 36.73%and 33.92%(P=0.577), and DCR1 were 79.59%and 89.29%(P=0.186);the ORR2 of G-T group and T-G group were 4.08%and 5.36%(P=0.085), and DCR2 were 51.02%and 66.07%, respectively (P=0.118). Hematologic toxicities was more frequent in G-T group, the patients experienced more grade 3-4 lower hemoglobin (P=0.027) and thrombocytopenia (P=0.002). Conclusion hTe effcacy of ifrst line gemcitabine+/-platinum followed by second line taxanes+/-platinum and the reverse sequence was similar, and the toxicities was tolerable. Both sequential patterns were effective in advanced squamous lung cancer.

Objective To detect time-dependent change of neuritin expression in brain tissues after traumatic brain injury and discuss the effect of neuritin after brain damage occurred. Methods Forty-two rats were divided into normal group, control and experimental group. According to the postoperative time divided into 6 subgroups, including 6 hours group, 12 hours group, 24 hours group, 3 days group, 7 days group and 14 days group. Immunohistochemical and western-blot were used to detected the protein expression levels of neuritin. Results The immunohistochemical staining indicated that the positive expression of neuritin was strong in the cytomembrane and cytoplasms of the neurons, with a higher intensity, 6 hours after the operation. 12 hours after the operation last to the seventh day, the neurons with the strongest positive expression, is significantly higher than control group and normal group, significant decrease on the fourteenth day. The result of western-blot indicated that the level of neuritin protein sharply increased at 6 hours, reached the peak on 24 hours and after lasted to the seventh day, significantly higher than control group and normal group (P < 0.01), significant decrease on the fourteenth day (P < 0.05). Conclusion Up-regulation of neuritin in cerebral contusion tissues may play an important role after traumatic brain injury.

Adult ; Aged ; Aged, 80 and over ; Carcinoma, Non-Small-Cell Lung ; genetics ; DNA Mutational Analysis ; methods ; DNA, Neoplasm ; genetics ; Female ; Humans ; Lung Neoplasms ; genetics ; Male ; Middle Aged ; Mutation ; Mutation Rate ; Receptor, Epidermal Growth Factor ; genetics

Objective To establish a serum-free primary culture method for cortical neurons of new-born rats .Methods The cortical tissue was digested and the cells were planted in the medium containing 10% fetal bovine serum ,and then maintained feed-ing with neurobasal medium containing B27 after 4 to 8 h .The morphology was observed under phase-contrast microscope .RT-PCR ,Western blot and immunocytochemistry were applied to identify the expression of NSE gene and protein in neurons .Results A large number of neurons began to adhere to the cover glasses after 2 to 8 h .They showed different shapes-shuttle ,triangle pyram-idal ,or no regular after clinging to the plate .Their processes connected to nets and were different in length and thickness .They well developed at the 7th to 10th day .The isolated and cultured cells were confirmed as neurons by RT-PCR ,Western blot and immuno-cytochemistry .Conclusion This technique is an easy and practical tool for primary culture of new-born rats cortical neurons with high purity ,and can be used as an in-vitro model of research .